PHYLOGENETIC STUDY OF GENUS SPATHOGLOTTIS BLUME (ORCHIDACEAE) IN MALESIA

FARAH ALIA NORDIN

UNIVERSITI SAINS MALAYSIA

2018 PHYLOGENETIC STUDY OF GENUS SPATHOGLOTTIS BLUME (ORCHIDACEAE) IN MALESIA

by

FARAH ALIA NORDIN

Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy

February 2018 ACKNOWLEDGEMENT

This thesis was made possible through the generosity of many individuals and agencies.

Firstly, sincere thanks to my supervisors, Professor Dr. Ahmad Sofiman bin Othman and Assoc. Prof. Dr. Kartini binti Saibeh for their guidance and patience throughout this study.

I would like to acknowledge Professor Dr. Rusea Go (Universiti Putra Malaysia, UPM), Dr. Hubert Kurzweil (Singapore Botanic Gardens), and Peter O’ Byrne who have provided excellent accounts for the alpha taxonomy for the genus studied.

To Forestry Department Peninsular Malaysia, Forest Department of Sarawak; notably Puan Nur Afiza binti Umar, Forest Department of Sabah, Mrs. C.Y. Chung of Sabah Biodiversity Centre, Ms. Nelly Majuakim from the Board of Trustees of the Sabah Parks; and Mr. Khairul Faizin and Mr. Mohd Sufian bin Abdul Kadir from Johor National Parks Corporation for providing the permits and export licenses required to collect the plants studied.

To Lab 409, School of Biological Sciences for providing the excellent laboratory facilities to carry out the molecular work.

To my collaborators Datuk Seri Lim Chong Keat (Folia Malaysiana Foundations), Mr. Reinheart Simarmata (Lembaga Ilmu Pengetahuan Indonesia, LIPI), Mr. Sahal Muadz (LIPI), Mr. Roy Banka (LAE Papua New Guinea National Herbarium), Mr. Handry Mujih (Kinabalu Park), Mr. Ong Poh Teck (Forest Research Institute of Malaysia, FRIM), Mrs. Suzana binti Sabran (Sabah Forestry Herbarium, SAN), Ms. Melly (UMS), Mr. Mohd Nasir (Terengganu Forestry Department), Mr. Ariek Firmansyah, Ms. Novianti, Mr. Kurt Keller, Mr. Lawrence, Mr. Rainol Sulutan, and Mr. Mohd Anuar bin Abdul Halim who provided me with their invaluable field observations and living samples for the plants studied in this genus.

To Mr. Mohd Shafreen bin Mad Isa of Department of Mineral and Geoscience Malaysia (Sabah) for granting permit access and has selflessly accompanied me to Mamut Copper Mine in Ranau.

To Dr. Richard Chung (FRIM Herbarium, KEP), Dr. Yong Kien Thai (Herbarium University of Malaya, KLU), Mr. Ahmad Damanhuri bin Mohamad (Herbarium Universiti Kebangsaan Malaysia, UKMB), Ms. Rimi binti Repin (Kinabalu National Park Herbarium), Dr. Rahmad bin Zakaria and Mr. Shunmugam (Herbarium of the School of Biological Sciences, USM), and Prof. Dr. Rusea Go (Herbarium of the Department of Biology, UPM) for granting me access to the herbaria to examine the historical specimens of the genus.

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To Dr. Sangmi Eum (Korea Research Institute of BioKorea Research Institute of Bioscience and Biotechnology, KRIBB), Prof. Niels Jacobsen (University of Copenhagen), Dr. Jayaraj Vijaya Kumaran (Universiti Malaysia Kelantan, UMK), and Mr. Mohd Akmal bin Mohd Raffi (Institute of Bioscience, UPM) for providing their ready guidance in data analysis.

To Mr. Wislee (Telupid District Forestry Office, Sabah), Mr. Mohd Anuar (Gurun Forestry and Ranger Office, Kedah), Mr. Samsu Bahari (Terengganu Forestry Department), Mr. Faizal bin Azmi, Mr. Azlan bin Azmi, Mr. Asri bin Azmi, and Mr. Azizuddin bin Abd. Hamid for guiding me braving through the wild, adventurous forests of Sabah, Terengganu, and Kedah.

To the staff of Biology Tropical and Conservation Insitute of Universiti Malaysia Sabah (UMS) for taking care of the living collections which have enabled our continuous observation of the living plants.

To my dear friends, who have been keeping up with me since the beginning and had restlessly assisting me in the fields and lab: Mr. Khairul Nasiruddin bin Abu Mangsor, Mr. Mohd Shukor, Mr. Nizam, Mr. Nazri, Ila, Ayun, Yanie, Radziah, Fahmi, Bard, Masyitah, Shakina, Fisya, Leen, Fasehah, Komala, Dr. Veera, Dr. Suganthi, Weenee, Adila, Siti Fatimah, Mike, Ikhwan, Vanielie, Edward, Fitri, Syuhada, Atiqah, Syikin, Faisal, Shi Yeu, Afifah, Najidah, Kaz, Ee, Vhenosha, Mr. Helmi, Safura, Odah, Azira, Rajiv, Fatin, Kak Ija, Abang Fisol, Abang Sukri, Abang Shaiful, Abang Azizul, Burhan, Firdaus, Pian, and Saufi; all of you are indeed beautiful and unselfish souls.

To Ministry of Higher Education and Universiti Sains Malaysia for the award of scholarships under the Skim Latihan Akademik Bumiputra (SLAB) and Academic Staff Training Scheme (ASTS) rewarded.

To my family, exclusively my mother, Emak; your prayers are my courage’s. Ento, Entin, Nona, Jahpen, Cha, Adik, Kak Sue, Kak Hani, Abg Fazil, Kak Watie, and Zul; you are my tremendous support system. I owe you all a lifetime.

To my late father, Abah; this thesis is specially dedicated for you.

Thank you.

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TABLE OF CONTENTS

Acknowledgement ii Table of Contents iv List of Tables ix List of Figures xi List of Plates xxv List of Abbreviations xxviii Abstrak xxx Abstract xxxiii

CHAPTER 1: INTRODUCTION 1.1 General Introduction 1 1.2 Objectives 5

CHAPTER 2: LITERATURE REVIEW 2.1 What and Where is Malesia? 7 2.1.1 Wallace’s Line – Two or Three Phytogeographical Areas? 10 2.1.2 The Demarcation and Internal Division of Malesia 11 2.2 Plate Tectonics and Changing Palaeogeography in Malesia 14 2.3 Plants Distribution Patterns and Floristic Exchange in Malesia 17 2.4 The Orchids Family 20 2.4.1 Diversification of the Orchidaceae 21 2.5 The Orchid Flower 24 2.6 The Genus Spathoglottis Blume 31 2.7 Revision Works on Genus Spathoglottis in Malesia 35 2.8 Distribution of Spathoglottis in Malesia 36 2.9 Revealing Species Relationships in Orchids Using Molecular Data 40 2.10 Phylogenetic Study and Molecular Data 41

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2.10.1 The Nuclear Ribosomal DNA (nrDNA) Genes 42 2.10.2 The Internal Transcribed Spacer as a Phylogenetic Marker 43 2.10.3 The Plastid Genome 44 2.10.4 The matK Plastid Gene 46 2.10.5 The Non•Coding trnL•F Intergenic Spacer 49

CHAPTER THREE: MORPHOLOGICAL AND ECOLOGICAL ANALYSES FOR THE RECOGNITION OF GROUPS WITHIN SPATHOGLOTTIS 3.1 Introduction 51 3.2 Materials and Methods 53 3.2.1 Taxon Sampling 53 3.2.2 Species Identification and Enumeration 53 3.2.3 Morphological Data 54 3.2.4 Character Matrix and Cladistic Analysis 55 3.3 Results and Discussion 56 3.3.1 Taxa Enumeration and Species Biogeographical Distribution 56 3.3.2 Proposed Island Groupings within Spathoglottis in Malesia 70 3.3.3 Vegetative and Reproductive Structures of Spathoglottis 71 3.3.4 Taxonomic Literature 74 3.3.5 Artificial Key to Species of Spathoglottis Blume 76 3.3.6 Ecology and Habitat Preferences 93 3.3.7 Character Matrix and Cladistic Analysis of Spathoglottis species 96 in Malesia 3.4 Conclusion 112

CHAPTER 4: MOLECULAR ANALYSIS 4.1 Introduction 113 4.2 Materials and Methods 114

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4.2.1 Plant Materials 114 4.2.2 DNA Extraction 118 4.2.2(a) CTAB Method 118 4.2.3 Polymerase Chain Reaction (PCR) of nrITS, matK, and trnL-F 121 4.2.3(a) PCR Primers 121 4.2.3(b) PCR Optimization and Amplification 123 4.2.3(c) PCR Product Purification 126 4.2.3(d) DNA Sequencing 127 4.2.3(e) Database Search – BLAST 127 4.2.4 Phylogenetic Analyses 128 4.2.4(a) Test of Incongruence 129 4.2.4(b) Maximum Likelihood Analysis 129 4.2.4(c) Maximum Parsimony Analysis 130 4.2.4(d) Bayesian Inference Analysis 130 4.3 Results 131 4.3.1 DNA Extraction and PCR Products of Spathoglottis Samples 132 4.3.2 Phylogenetic Analysis based on Internal Transcribed Spacer 135 Region of the Nuclear Ribosomal Gene 4.3.3 Phylogenetic Analysis based on matK Region 144 4.3.4 Phylogenetic Analysis based on trnL-F Region 152 4.3.5 Combined Molecular Data Analysis 161 4.3.5(a) Analysis of Combined Plastid Sequence Data 161 4.3.5(b) Combined Plastid and nrITS Data 169 4.4 Discussion 178 4.4.1 Monophyly of Four Complexes of Genus Spathoglottis 180 4.4.2 Comparison between Molecular and Morphological Study 184 4.4.3 Proposed Taxonomic and Nomenclatural Changes for 188 Spathoglottis plicata var. alba and Further Investigation Work for S. kimballiana 4.5 Conclusion 190

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CHAPTER 5: THE YELLOW SPATHOGLOTTIS COMPLEX: TAXONOMIC POSITION OF THE NARROW-LIP SPATHOGLOTTIS 5.1 Introduction 192 5.2 Materials and Methods 197 5.2.1 Macro-, Micromorphological and Ecological Analyses 197 5.2.2 Molecular Analyses 199 5.2.3 DNA Barcoding Gap Analysis 199 5.3 Results 201 5.3.1 Morphological and Ecological Assessments of the Yellow 201 Spathoglottis Complex 5.3.2 Molecular Analyses 214 5.3.2(a) Phylogenetic Analysis based on nrITS Sequences 214 5.3.2(b) Phylogenetic Analysis based on matK Region 222 5.3.2(c) Phylogenetic Analysis based on trnL-F Region 228 5.3.2(d) Analysis of Combined Plastid Sequence Data 235 5.3.2(e) Combined Plastid and nrITS Data 245 5.3.3 Species Delimitation based on the Barcoding Gap Analysis 253 5.4 Discussion 266 5.4.1 Delimitation of species between Spathoglottis aurea and 270 S. microchilina based on Molecular and Morphological Study 5.5 Conclusion 272

CHAPTER 6: BIOGEOGRAPHY AND CHARACTER EVOLUTION 6.1 Introduction 273 6.2 Materials and Methods 275 6.3 Results 276 6.3.1 Hypotheses on Character Evolution of Genus Spathoglottis 276 based on Homologous and Homoplastic Characters

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6.3.2 Hypotheses on Character Evolution within the Yellow 312 Spathoglottis Complex (YSC) based on Homologous and Homoplastic Characters 6.3.3 Geographical Distribution of Genus Spathoglottis in Malesia 328 6.3.4 Plate Tectonic Movements and the Proposed Five Island 328 Groupings 6.4 Discussion 334 6.4.1 Character Evolution in Genus Spathoglottis 334 6.4.2 Geographical Distribution of Genus Spathoglottis in Malesia, 337 Plate Tectonic Movements and the Proposed Five Island Groupings 6.5 Conclusion 347

CHAPTER 7: SUMMARY, GENERAL CONCLUSION AND 348 RECOMMENDATIONS FOR FUTURE RESEARCH

REFERENCES 354 APPENDICES LIST OF PUBLICATIONS

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LIST OF TABLES

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Table 2.1 Circumscription of genus Spathoglottis in tribe Collabieae 34 based on treatments by various authors.

Table 2.2 List of Spathoglottis species in Malesia and their area of 37 occurrence based on botanical country.

Table 3.1 Checklist of species/infraspecific taxa collected in this 57 study.

Table 3.2 Species list, collection localities, and herbarium voucher 58 numbers of the species used in this study. GPS coordinates are not available for locations denoted with asterisk (*).

Table 3.3 Vegetative characters and its character states. 96

Table 3.4 Reproductive characters and its character states. 97

Table 3.5 Ecological characters and its character states. 98

Table 3.6 Distribution of character states among taxa for the 72 99 morphological characters and three ecological characters. A symbol ‘?’ denotes for missing or not applicable character.

Table 4.1 Species list, herbarium voucher numbers, and GeneBank 116 accession numbers of the Spathoglottis and outgroup species used in this study.

Table 4.2 Primers information for polymerase chain reaction 122 amplification of nrITS, matK, and trnL-F plastid gene.

Table 4.3 PCR cycling profiles for the amplification of ITS, matK 124 and trnL-F genes.

Table 4.4 Contents of 50µL reaction mixtures for PCR amplification 125 of ITS, matK and trnL-F gene.

Table 4.5 Sequence data analysis of ITS 1, 5.8S and ITS 2 regions in 136 12 species and three varieties of Spathoglottis

Table 5.1 Species list, herbarium voucher numbers, and GenBank 198 accession numbers of the 29 individuals of the Yellow

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Spathoglottis Complex and two outgroup species used in this study.

Table 5.2 Sequence data analysis of ITS 1, 5.8S and ITS 2 regions in 215 three species and two varieties of the Yellow Spathoglottis Complex.

Table 5.3 Analysis of intra-specific variation and inter-specific 255 divergence of the ITS 2 sequences in 12 species and three varieties of Spathoglottis (including species of the YSC).

Table 5.4 The intra-/inter-specific divergence (%) between species 257 within the YSC based on ITS 2 sequences.

Table 5.5 Analysis of intra-specific variation and inter-specific 257 divergence of the matK sequences in 12 species and three varieties of Spathoglottis (including species of the YSC).

Table 5.6 The intra- and inter-specific divergence (%) between 258 species within the YSC based on matK sequences.

Table 5.7 Analysis of intra-specific variation and inter-specific 260 divergence of the trnL-F sequences in 12 species and three varieties of Spathoglottis (including species of the YSC).

Table 5.8 The intra- and inter-specific divergence (%) between 262 species within the YSC based on trnL-F sequences.

Table 5.9 Analysis of intra-specific variation and inter-specific 263 divergence of the combined plastid and nuclear sequences in 12 species and three varieties of Spathoglottis (including species of the YSC).

Table 5.10 The intra- and inter-specific divergence (%) between 264 species within the YSC based on combined plastid and nuclear sequences.

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LIST OF FIGURES

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Figure 2.1 Map of the boundaries in Malesian floristic region as 8 recognized by Zollinger in 1857. Total grey area is in the widest sense and the dark•grey area in more restricted sense. New Guinea is largely excluded. The delimitation by Van Steenis (1948; 1950) is marked by three demarcation knots (red circle). The numbers indicate the number of plant genera not crossing the knots. The different lines indicate Wallace’s Line and its variants by different authors that split up Malesia to the eastern and western parts. Map adopted from Raes and Van Welzen (2009).

Figure 2.2 Map of Malesia. Re-draw from Van Welzen et al. (2011). 12

Figure 2.3 Three phytogeographical subunits in Malesia: Sunda, Sahul 14 and Wallacea. The area of endemism employed and intercontinental dispersal dynamics. Map adopted from Crayn et al. (2014).

Figure 2.4 Relationship between the five orchid subfamilies based on 30 Chase et al., (2015).

Figure 2.5 Distribution of Spathoglottis corresponding to the seven 39 island groupings: (1) Indo-China, (2) Malay Peninsula + Sumatra (Sunda Shelf), (3) Borneo (Sunda Shelf), (4) Java (Wallacea), (5) Sulawesi (Wallacea), (6) the Philippines (Wallacea) and (7) New Guinea (Sahul Shelf). Spathoglottis plicata is a widespread species throughout Malesia.

Figure 2.6 Organization of the three subunits of nuclear ribosomal 43 DNA. The large subunits of rDNA (18S and 26S) are separated by the internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) respectively. The ITS1 and ITS2 regions are separated by the small subunit rDNA, 5.8S.

Figure 2.7 Gene map of Dioscorea elephantipes plastid genome. 45 Thick lines represent the extent of the inverted repeats; separating the large single-copy region (LSC) from the small single-copy region (SSC). Genes shown outside the circle are transcribed in the clockwise direction, and those on the inside of the circle are transcribed in the counter-

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clockwise direction. Genes containing introns are with asterisks. Map adopted from Hansen et al., 2007.

Figure 2.8 Structure of matK gene. 47

Figure 2.9 The position of the three non•coding regions of plastid 50 DNA (trnT-trnL intron; trnL intron; trnL-F intron).

Figure 3.1 A majority rule consensus of 100 equally parsimonious 107 trees of Spathoglottis species based on morphological sequence data. Tree length = 251; CI = 0.51; RI = 0.54. Groups I and II denote for the supergroups Purple Spathoglottis Complex and Yellow Spathoglottis Complex, respectively. Numbers at nodes denote for the informal groups in Spathoglottis.

Figure 4.1 DNA with good quality and quantity were obtained using 132 the CTAB method (Doyle and Doyle, 1987) with some modifications. DNA sample isolated from four Spathoglottis species were electrophorised on 0.8% (w/v) agarose gel at 90 V. Lane M: Lambda DNA/HindIII Marker; Lane 1: Spathoglottis aurea FAN035; Lane 2: S. aurea FAN040; Lane 3: S. kimballiana var. kimballiana FAN067; Lane 4: S. affinis FAN028.

Figure 4.2 The SEVAG solutions [chloroform-isoamyl alcohol (24: 133 1)] from a sample of Spathoglottis aurea that contains the aqueous layers of supernatant and polysaccharides debris.

Figure 4.3(a) The banding patterns produced by amplification of ITS 134 gene using primer set 17SE and 26SE on 1.8% agarose gel electrophoresis. [MgCl2]= 2.0 mM, annealing temperature = 58°C. Lane M: 1 kb DNA Ladder; Lane 1: FAN067 (S. kimballiana var. kimballiana); Lane 2: FAN068 (S. pubescens); Lane 3 and 4: FAN020 and FAN023 (S. affinis); Lane 5 and 6: FAN076 and FAN077 (S. kimballiana var. angustifolia); Lane 7: FAN093 (S. kimballiana var. kimballiana).

Figure 4.3(b) The banding patterns produced by amplification of trnL-F 134 region using primer set c and f on 1.8% agarose gel electrophoresis. [MgCl2]= 2.5 mM, annealing temperature = 56°C. Lane M: 1 kb DNA Ladder; Lane 1: FAN094 (S. gracilis); Lane 2: FAN092 (S. aurea); Lane 3: FAN038 (S. aurea); Lane 4: FAN016 (S. hardingiana).

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Figure 4.3(c) The banding patterns produced by amplification of matK 135 gene using primer set 360F and 1326R on 1.8% agarose gel electrophoresis. [MgCl2]= 2.0 mM, annealing temperature = 48°C. Lane M: 1 kb DNA Ladder; Lane 1, 2, 4, 5, 6: FAN017, FAN050, FAN011, FAN033, FAN015 (S. plicata); Lane 3: FAN034 (S. plicata var. alba).

Figure 4.4 The majority-rule consensus tree results from Maximum 138 Likelihood analysis (ML) of genus Spathoglottis based on ITS sequences dataset. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-2833.810). Clade II-IV shows the ingroups of genus Spathoglottis; Clade I indicates outgroup. Clade II and III are the Dwarf Spathoglottis complexes, while Clade IV (A and B) consists of the Large Spathoglottis species. Clade B is further separated into subclades B1 and B2; each holds the Large Purple and Large Yellow Spathoglottis Complex, respectively. Taxa highlighted in yellow box are the yellow flower Spathoglottis while purple box comprises of flowers in different shades of purple. Groupings of taxa were also based on the shape of the labellum; either narrow or broad/bilobulate.

Figure 4.5 The consensus tree inferred from seven equally 139 parsimonious trees via Maximum Parsimony analysis (MP) of genus Spathoglottis based on ITS sequences. Branches corresponding to partitions reproduced in less than 50% are collapsed. Tree length= 346; consistency index, CI= 0.712; retention index, RI= 0.909; and composite index, RC= 0.699 (RC= 0.647) for all sites and parsimony-informative sites (in parentheses). Clade II-IV shows the ingroups of genus Spathoglottis; Clade I indicates outgroup. Clade II and III are the Dwarf Spathoglottis complexes, while Clade IV (A and B) consists of the Large Spathoglottis species. Clade B is further separated into subclades B1 and B2; each holds the Large Purple and Large Yellow Spathoglottis Complex, respectively. Taxa highlighted in yellow box are the yellow flower Spathoglottis while purple box comprises of flowers in different shades of purple.

Figure 4.6 The optimal tree resulting from Bayesian Inference (BI) of 141 genus Spathoglottis based on ITS sequences. Values on the nodes correspond to posterior probabilities (PP). Clade II-

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IV shows the ingroups of genus Spathoglottis; Clade I indicates outgroup. Clade II and III are the Dwarf Spathoglottis complexes, while Clade IV (A and B) consists of the Large Spathoglottis species. Clade IV-B is further separated into subclades B1 and B2; each holds the Large Purple and Large Yellow Spathoglottis Complex, respectively. Taxa highlighted in yellow box are the yellow flower Spathoglottis while purple box comprises of flowers in different shades of purple. Groupings of taxa were also based on the shape of the labellum; either narrow or broad/bilobulate.

Figure 4.7 The majority-rule consensus tree results from Maximum 146 Likelihood analysis (ML) of genus Spathoglottis based on matK sequence data. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-1845.955). Clade II and III holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. Clade II comprises of the Dwarf Spathoglottis complexes, while Clade III consists of the Large Spathoglottis species. Clade III is further separated into subclades A and B; the Large Purple and Large Yellow Spathoglottis Complex, respectively. Taxa highlighted in yellow box are the yellow flower Spathoglottis while purple box comprises of flowers in different shades of purple. Groupings of taxa were also based on the shape of the labellum; either narrow or broad/bilobulate. Both clades of Dwarf and Large Spathoglottis are monophyletic.

Figure 4.8 The consensus tree inferred from four equally 148 parsimonious trees via Maximum Parsimony analysis (MP) of genus Spathoglottis based on matK sequence data. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 94; consistency index, CI= 0.746; retention index, RI= 0.943; and composite index, RC= 0.783 (0.704) for all sites and parsimony-informative sites (in parentheses). Clade II and III holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. Clade II comprises of the Dwarf Spathoglottis complexes, while Clade III consists of the Large Spathoglottis species. Clade III is further separated into subclades A and B; the Large Purple and Large Yellow Spathoglottis Complex, respectively.

Figure 4.9 The optimal tree resulting from Bayesian Inference (BI) of 149 genus Spathoglottis based on matK sequence data. Values

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on the nodes correspond to posterior probabilities (PP). Both Dwarf Spathoglottis (Clade II) and Large Spathoglottis (Clade III) are monophyletic. Independently, the Large Purple Spathoglottis Complex is polyphyletic.

Figure 4.10 The majority-rule consensus tree results from Maximum 155 Likelihood analysis (ML) of genus Spathoglottis based on TrnL-F region. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-2732.540). Clade II and III holds the Dwarf Spathoglottis complexes while Clade IV and V consists of the Large Spathoglottis species; and Clade I indicates outgroups. Independently, the Large Yellow Spathoglottis is monophyletic, while the Large Purple Spathoglottis is polyphyletic.

Figure 4.11 The consensus tree inferred from six equally parsimonious 156 trees via Maximum Parsimony analysis (MP) of genus Spathoglottis based on TrnL-F region. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 267; consistency index, CI= 0.723; retention index, RI= 0.902; and composite index, RC= 0.719 (0.652) for all sites and parsimony- informative sites (in parentheses). Clade II and III holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. Clade III comprises of the Dwarf Spathoglottis complexes (Subclade III-A) and the Large Yellow Spathoglottis Complex (Subclade III-B). Separately, the Large Purple Spathoglottis Complex (Clade II) formed a sister clade to all Spathoglottis species at the basal of the tree.

Figure 4.12 The optimal tree resulting from Bayesian Inference (BI) of 158 genus Spathoglottis based on TrnL-F region. Values on the nodes correspond to posterior probabilities (PP). Clade II and III shows the ingroups of genus Spathoglottis; Clade I indicates outgroup. Both Dwarf Yellow and Dwarf Purple Spathoglottis complexes (Subclade III-A) nested within Clade III, as sister to the Large Yellow Spathoglottis Complex (Subclade III-B). The Large Purple Spathoglottis Complex formed Clade II at the basal of the tree.

Figure 4.13 The majority-rule consensus tree results from Maximum 164 Likelihood analysis (ML) of genus Spathoglottis based on combined plastid gene dataset. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-4503.460). Clade II and III

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holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. Clade II comprises of the Dwarf Spathoglottis complexes, while Clade III consists of the Large Spathoglottis species. Clade III is further separated into subclades A and B; the Large Purple and Large Yellow Spathoglottis Complex, respectively. Taxa highlighted in yellow box are the yellow flower Spathoglottis while purple box comprises of flowers in different shades of purple. Groupings of taxa were also based on the shape of the labellum; either narrow or broad/bilobulate. Both clades of Dwarf and Large Spathoglottis (Clade II and Clade III) are monophyletic.

Figure 4.14 The consensus tree inferred from eight equally 165 parsimonious trees via Maximum Parsimony analysis (MP) of genus Spathoglottis based on combined plastid gene dataset. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 337; consistency index, CI= 0.780; retention index, RI= 0.935; and composite index, RC= 0.799 (0.729) for all sites and parsimony-informative sites (in parentheses). Clade II and III holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. Clade II comprises of the Dwarf Spathoglottis complexes, while Clade III (A and B) consists of the Large Spathoglottis species. Both Clade II and III are monophyletic.

Figure 4.15 The optimal tree resulting from Bayesian Inference (BI) of 167 genus Spathoglottis based on combined plastid gene dataset. Values on the nodes correspond to posterior probabilities (PP). Both Dwarf Spathoglottis (Clade II) and Large Spathoglottis (Clade III) are monophyletic. Independently, the Large Yellow Spathoglottis Complex (Subclade III-B) is monophyletic while the Large Purple Spathoglottis Complex (Subclade III-A) is polyphyletic.

Figure 4.16 The majority-rule consensus tree results from Maximum 172 Likelihood analysis (ML) of genus Spathoglottis based on combined plastid and nuclear gene dataset. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-7663.795). Clade II-IV holds all 15 species (ingroups) of genus Spathoglottis; Clade I indicates outgroup. The Dwarf Yellow Spathoglottis (Clade II) formed a basal group; while the Dwarf Purple Spathoglottis (Clade III) is sister to all Large Spathoglottis species. Clade IV that comprises of the Large Spathoglottis complexes is monophyletic.

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Independently, the Large Yellow Spathoglottis Complex (Subclade IV-B) is monophyletic while the Large Purple Spathoglottis Complex is polyphyletic.

Figure 4.17 The consensus tree inferred from two equally parsimonious 173 trees via Maximum Parsimony analysis (MP) of genus Spathoglottis based on combined plastid and nuclear gene dataset. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 695; consistency index, CI= 0.709; retention index, RI= 0.906; and composite index, RC= 0.711 (0.643) for all sites and parsimony-informative sites (in parentheses). Clade I indicates outgroup. Clade II comprises of the Dwarf Yellow Spathoglottis Complex, as sister to all Spathoglottis species. Clade III holds the Dwarf Purple Spathoglottis Complex (Subclade III-A), Large Purple Spathoglottis Complex (Subclade III-B1) and Large Yellow Spathoglottis Complex (Subclade III-B2). The Large Spathoglottis group (Clade III-B) is monophyletic.

Figure 4.18 The optimal tree resulting from Bayesian Inference (BI) of 175 genus Spathoglottis based on combined plastid and nuclear gene dataset. Values on the nodes correspond to posterior probabilities (PP). The Large Spathoglottis (Clade IV) is monophyletic. Independently, the Large Yellow Spathoglottis Complex (Subclade IV-B) is monophyletic, while the Large Purple Spathoglottis Complex is polyphyletic. The Dwarf Yellow Spathoglottis (Clade II) is sister to all Spathoglottis species.

Figure 4.19 A majority rule consensus of 100 equally parsimonious 185 trees of Spathoglottis species based on morphological sequence data. Tree length= 239. Clade I= S. unguiculata (the Short-Column Spathoglottis); Clade II= S. parviflora (the Oblong Spathoglottis); Clade III; Clade III-A= Large Purple Spathoglottis Complex (Purple Spathoglottis Complex); Clade III-B= Yellow Spathoglottis Complex; Clade III-B1= Dwarf Yellow Spathoglottis Complex; Clade III-B2= Large Yellow Spathoglottis Complex; Subclade III-B3= the Narrow-lip Spathoglottis; and Subclade III-B4= the Broad-lip/Bilobulate Spathoglottis. Notes: Purple box indicates Purple Spathoglottis Complex, while yellow box indicates Yellow Spathoglottis Complex.

Figure 5.1 A majority rule consensus of 100 equally parsimonious 212 trees of the yellow Spathoglottis species. Clade I denotes for the Narrow-Lip Spathoglottis, while Clade B consists

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of species with broad-lip/bilobulate midlobe.

Figure 5.2 The majority-rule consensus tree results from Maximum 217 Likelihood analysis (ML) of ITS sequence dataset of Yellow Spathoglottis Complex. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-1673.476). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicates outgroup. Clade III is monophyletic, comprises of all montane Spathoglottis species. A riverine/lowland species, S. kimballiana var. angustifolia formed a basal clade in Clade II. Spathoglottis aurea and S. microchilina, members of the Narrow-lip Spathoglottis were grouped separately in Subclades III-B1, III-B3, and III-B4.

Figure 5.3 The consensus tree inferred from ten equally parsimonious 218 trees via Maximum Parsimony analysis (MP) of Yellow Spathoglottis Complex based on ITS sequence. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 110; consistency index, CI= 0.846; retention index, RI= 0.927; and composite index, RC= 0.801 (0.785) for all sites and parsimony- informative sites (in parentheses). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicates outgroup. Clade III is monophyletic, comprises of all montane Spathoglottis species. A riverine/lowland species, S. kimballiana var. angustifolia formed a basal clade in Clade II. Spathoglottis aurea and S. microchilina, members of the Narrow-lip Spathoglottis were grouped separately in Subclades III-B1, III-B3, and III-B4.

Figure 5.4 The optimal tree resulting from Bayesian Inference (BI) of 220 Yellow Spathoglottis Complex based on ITS gene. Values on the nodes correspond to posterior probabilities (PP). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicates outgroup. Clade III is monophyletic, comprises of all montane Spathoglottis species. A riverine/lowland species, S. kimballiana var. angustifolia formed a basal clade in Clade II. Spathoglottis aurea and S. microchilina, members of the Narrow-lip Spathoglottis were grouped separately in Subclades III-A, III-B, and III-E.

Figure 5.5 The majority-rule consensus tree results from Maximum 224 Likelihood analysis (ML) of Yellow Spathoglottis Complex based on matK sequence data. Numbers at nodes

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represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-1421.990). Clade II-V consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. The Grassy Spathoglottis complexes (Clade III and IV) formed polytomies on the tree spine. Spathoglottis aurea and S. microchilina, members of the Narrow-lip Spathoglottis were grouped separately in Clade II and V.

Figure 5.6 The consensus tree inferred from four equally 225 parsimonious trees via Maximum Parsimony analysis (MP) of Yellow Spathoglottis Complex based on matK sequence data. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 31; consistency index, CI= 0.892; retention index, RI= 0.971; and composite index, RC= 0.877 (0.867) for all sites and parsimony-informative sites (in parentheses). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicates outgroup. Clade III is monophyletic; comprises of all montane and Plicate Spathoglottis species. The Grassy Spathoglottis complex formed Clade II at the basal of the tree, and is also monophyletic. In separate groupings, Clade III (A and B) hold together both Spathoglottis aurea and S. microchilina; members of the Narrow-lip Spathoglottis.

Figure 5.7 The optimal tree resulting from Bayesian Inference (BI) of 226 Yellow Spathoglottis Complex based on matK sequence data. Values on the nodes correspond to posterior probabilities (PP). Clade II-V consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. The Grassy Spathoglottis complexes (Clade IV and V) formed polytomies on the tree spine. Spathoglottis aurea and S. microchilina; members of the Narrow-lip Spathoglottis were grouped separately in Clade II and III, respectively.

Figure 5.8 The majority-rule consensus tree results from Maximum 230 Likelihood analysis (ML) of Yellow Spathoglottis Complex based on TrnL-F region. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-2066.595). Clade II-IV consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. Spathoglottis microchilina is nested within Clade III with S. gracilis and S. kimballiana var. angustifolia, while all S. aurea is grouped together in Clade IV.

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Figure 5.9 The consensus tree inferred from ten equally parsimonious 232 trees via Maximum Parsimony analysis (MP) of Yellow Spathoglottis Complex based on TrnL-F region. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 102; consistency index, CI= 0.970; retention index, RI= 0.989; and composite index, RC= 0.960 (0.960) for all sites and parsimony- informative sites (in parentheses). Clade II-V consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. Clade V is monophyletic; holding both Spathoglottis microchilina and S. aurea (Narrow-lip Spathoglottis) in subclades V-A and V-B, respectively.

Figure 5.10 The optimal tree resulting from Bayesian Inference (BI) of 233 Yellow Spathoglottis Complex based on TrnL-F region. Values on the nodes correspond to posterior probabilities (PP). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. Spathoglottis microchilina is nested within Clade II with S. gracilis and the Grassy Spathoglottis, while all S. aurea is grouped together in Clade III.

Figure 5.11 The majority-rule consensus tree results from Maximum 239 Likelihood analysis (ML) of Yellow Spathoglottis Complex based on combined plastid gene dataset. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-3576.682). Clade II-V consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. The Grassy Spathoglottis complex (Clade II) is sister to all Plicate Spathoglottis. The Narrow-lip Spathoglottis complexes that comprise of S. microchilina and S. aurea are grouped together in Clade IV and V, respectively.

Figure 5.12 The consensus tree inferred from ten equally parsimonious 240 trees via Maximum Parsimony analysis (MP) of Yellow Spathoglottis Complex based on combined plastid gene dataset. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 142; consistency index, CI= 0.924; retention index, RI= 0.975; and composite index, RC= 0.907 (0.902) for all sites and parsimony-informative sites (in parentheses). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. Spathoglottis gracilis (Clade II) is sister to all yellow-flower Spathoglottis. The Grassy Spathoglottis species (Subclade III-A) embedded within Clade III; as sister to the Narrow-lip Spathoglottis

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complexes (Subclade III-B).

Figure 5.13 The optimal tree resulting from Bayesian Inference (BI) of 242 Yellow Spathoglottis Complex based on combined plastid gene dataset. Values on the nodes correspond to posterior probabilities (PP). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. The Grassy Spathoglottis complex (Clade II) is sister to all Plicate Spathoglottis. Clade III and Subclade III-B are monophyletic. Spathoglottis gracilis (Clade III-A) is sister to the Narrow-lip Spathoglottis. The Narrow-lip Spathoglottis complexes that comprise of S. microchilina and S. aurea are grouped together within Subclade III-B (B1 and B2, respectively).

Figure 5.14 The majority-rule consensus tree results from Maximum 247 Likelihood analysis (ML) of Yellow Spathoglottis Complex based on combined plastid and nuclear gene dataset. Numbers at nodes represent percent recovery in bootstrap analysis (1000 replicates) with highest log likelihood (-5445.757). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. A lowland/riverine species, Spathoglottis kimballiana var. angustifolia (Clade II) is sister to all yellow-flower Spathoglottis. Clade III is further divided into subclades A and B; which S. gracilis (Subclade III-A) is sister to all montane yellow-flower Spathoglottis. The Narrow-lip Spathoglottis complexes are nested together within Subclade III-B, separated by S. kimballiana var. kimballiana.

Figure 5.15 The consensus tree inferred from ten equally parsimonious 249 trees via Maximum Parsimony analysis (MP) of Yellow Spathoglottis Complex based on combined plastid and nuclear gene dataset. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. Tree length= 254; consistency index, CI= 0.872; retention index, RI= 0.948; and composite index, RC= 0.836 (0.827) for all sites and parsimony-informative sites (in parentheses). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. A lowland/riverine species, Spathoglottis kimballiana var. angustifolia (Clade II) is sister to all yellow-flower Spathoglottis. Clade III is further divided into subclades A and B; which S. gracilis (Subclade III-A) is sister to all montane yellow-flower Spathoglottis. The Narrow-lip Spathoglottis complexes are nested together within

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Subclade III-B (B1 and B2).

Figure 5.16 The optimal tree resulting from Bayesian Inference (BI) of 250 Yellow Spathoglottis Complex based on combined plastid and nuclear gene dataset. Values on the nodes correspond to posterior probabilities (PP). Clade II and III consists of all Large Yellow Spathoglottis species, while Clade I indicate outgroup. A lowland/riverine species, Spathoglottis kimballiana var. angustifolia (Clade II) is sister to all yellow-flower Spathoglottis. Clade III that holds all montane yellow-flower Spathoglottis is further divided into subclades A and B (B1 and B2). The Narrow- lip Spathoglottis complexes are grouped separately in Subclades III-B1 and III-B2.

Figure 5.17 Relative distribution of inter-specific divergence between 256 congeneric Spathoglottis species and intra-specific variation in the ITS2 region.

Figure 5.18 Relative distribution of inter-specific divergence between 259 congeneric Spathoglottis species and intra-specific variation in the matK region.

Figure 5.19 Relative distribution of inter-specific divergence between 261 congeneric Spathoglottis species and intra-specific variation in the TrnL-F region.

Figure 5.20 Relative distribution of inter-specific divergence between 265 congeneric Spathoglottis species and intra-specific variation in the combined plastid and nuclear gene region.

Figure 6.1 Tree A. Plant size; Tree B. Pseudobulb shape. 277

Figure 6.2 Tree A. Number of leaves per pseudobulb; Tree B. Leaf 279 colouration.

Figure 6.3 Tree A. Inflorescence ornamentation; Tree B. Sterile bract 281 shape.

Figure 6.4 Tree A. Floral bract senescence; Tree B. Floral bract shape. 283

Figure 6.5 Tree A. Floral bract attachment; Tree B. Floral bract 285 texture.

Figure 6.6 Tree A. Pedicel ornamentation; Tree B. Ovary swelling. 287

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Figure 6.7 Tree A. Number of flower per rachis; Tree B. Flower 289 colour.

Figure 6.8 Tree A. Flower resupination; Tree B. Flower pollination 291 strategy.

Figure 6.9 Tree A. Sepals and petals size; Tree B. Sepal 293 ornamentation.

Figure 6.10 Tree A. Lip (labellum) shape; Tree B. Width of midlobe. 296

Figure 6.11 Tree A. Lip ornamentation; Tree B. Sidelobe shape. 298

Figure 6.12 Tree A. Lip claw; Tree B. Auricles (teeth of lip). 300

Figure 6.13 Tree A. Callus shape; Tree B. Callus architecture. 302

Figure 6.14 Tree A. Column architecture; Tree B. Column size. 304

Figure 6.15 Tree A. Column cap shape; Tree B. Habit. 307

Figure 6.16 Tree A. Ecological niches; Tree B. Geology. 311

Figure 6.17 Tree A. Leaf vernation; Tree B. Leaf width. 313

Figure 6.18 Tree A. Flower size; Tree B. Shades of yellow. 315

Figure 6.19 Tree A. Flower pollination strategy; Tree B. Fruit set 318 percentage.

Figure 6.20 Tree A. Sepal colouration; Tree B. Lip (labellum) shape. 320

Figure 6.21 Tree A. Lip size; Tree B. Lip apex. 322

Figure 6.22 Tree A. Sidelobe length; Tree B. Sidelobe width. 323

Figure 6.23 Tree A. Callus architecture; Tree B. Column architecture. 325

Figure 6.24 Tree A. Ecological niches; Tree B. Geology. 327

Figure 6.25 The geographical distribution of genus Spathoglottis 329 throughout Malesia from a Maximum Likelihood analysis based on combined plastid and nuclear gene dataset. Grouping of the clades and subclades are corresponding to their particular island groupings.

Figure 6.26 Geographical distribution of genus Spathoglottis 331

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throughout Malesia based on their particular island groupings.

Figure 6.27 The geographical distribution of Yellow Spathoglottis 332 Complex throughout Malesia from a Maximum Likelihood analysis based on combined plastid and nuclear gene dataset. Grouping of the clades and subclades are corresponding to their particular island groupings.

Figure 6.28 Geographical distribution of Yellow Spathoglottis 333 Complex throughout Malesia based on their particular island groupings.

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LIST OF PLATES

Page

Plate 2.1(A•F) Epiphytic orchids (A) Agrostophyllum majus, (B) 23 Grammatophyllum speciosum, (C) Dendrobium hasseltti; lithophytic orchid (D) Ludisia discolor; and terrestrial orchids (E) Anoectochilus geniculatus, (F) Corybas holttumii in their natural habitat. (Pictures not following scale).

Plate 2.2(A•B) The flower structure of an orchid. (A) Arundina 26 graminifolia; (B) Paphiopedilum lowii.

Plate 2.3(A•F) Various ornaments of the labellum. (A) Keels on 28 Coelogyne xyrekes, (B) raised ridges/keels on Coelogyne pulverula, (C) papillae on Coelogyne tomentosa, (D) hairs on the lip of Dipodium pictum, (E) callus on Spathoglottis aurea, and (F) appandages of Anoectochilus geniculatus. (Pictures not following scale).

Plate 2.4 Column of Spathoglottis aurea with lip removed; side 29 view.

Plate 2.5(A•M) Spathoglottis plicata. (A) Habit, (B) flower, (C) 33 labellum, (D) dorsal sepal, (E) lateral sepal, (F) lateral petal, (G) column, dorsal view, (H) anther, (I) calli; dorsal view, (J) calli; side view, (K) pollinaria, (L) pollinia, and (M) fruits.

Plate 3.1 S. hardingiana (K20160013) growing on damp rocks, 66 in•situ Langkawi (Photo: Imin Kamin and Ong, P.T.).

Plate 3.2(A•C) Vegetative structures of S. plicata; (A) habit, (B) details 72 on the plicate leaf, and (C) pseudobulb and roots.

Plate 3.3(A•C) Reproductive structures of Spathoglottis species. (A) 73 Rachis and fruits of S. plicata, (B) flower parts of S. plicata, and (C) flower parts of S. aurea. (Pictures do not following scale).

Plate 3.4(A•C) S. hardingiana (FAN056), in situ Baling, Kedah. (A) 82 Habit, (B) flower; front view (C) flower; side view.

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Plate 3.5(A•D) S. pubescens (FAN068). (A) Habit, (B) flower; front 83 view, (C) flower; side view, (D) flower, back view.

Plate 3.6(A•B) S. eburnea (FAN022). (A) Flower; front view, (B) the 84 distinct white•coloured pseudobulb.

Plate 3.7(A•D) S. affinis (FAN028). (A) Habit, (B) flower; front view, 85 (C) flower; side view, (D) flower; back view.

Plate 3.8(A•D) S. lobbii (FAN025). (A) Habit, (B) flower; front view, 86 (C) flower; side view, (D) a swelling ovary.

Plate 3.9(A•D) S. microchilina (FAN083). (A) Habit, (B) pseudobulb, 87 (C) part of the inflorescence, (D) flower; front view.

Plate 3.10(A•D) S. aurea (FAN044). (A) Habit, (B) flower; front view, 88 (B) flower; side view.

Plate 3.11(A•D) (A) S. kimballiana var. kimballiana (FAN093); flower, 89 (B) S. kimballiana var. angustifolia (FAN076); habit, (C) S. kimballiana var. angustifolia flower; front view, (D) flower; side view. (C•D) photos courtesy of Peter O’Byrne, (KIP1266ff).

Plate 3.12(A•D) (A) S. kimballiana (FAN084); habit, (B) flower; front 90 view, (C) S. gracilis; habit, (D) flower; front and side view. (B) photo courtesy of Robert, A. Copyright 2015. (C­D) photos courtesy of Peter O’Byrne, (THH13­6­99).

Plate 3.13(A•C) S. parviflora (FAN061). (A) habit, (B) part of the 91 inflorescence (C) flower; front view.

Plate 3.14(A•E) (A•B) S. plicata var. alba (FAN034), flowers. (C) S. 92 unguiculata (FAN024); habit, (D) part of the inflorescence (E) flower; front view.

Plate 3.15(A•I) Sidelobes (Character 57) of (A) S. microchilina, (B) S. 101 aurea, (C) S. hardingiana, (D) S. plicata, (E) S. affinis, (F) S. lobbii, (G) S. kimballiana var. kimballiana, (H) S. kimballiana var. angustifolia, and (I) S. gracilis. Note: (H) Picture courtesy of Peter O’ Byrne.

Plate 3.16(A•I) Lip (Character 49) of (A) S. microchilina, (B) S. aurea, 102 (C) S. unguiculata, (D) S. affinis, (E) S. lobbii, (F) S. eburnea, (G•H) S. plicata, (I) S. plicata var. alba, and (I) S. parviflora.

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Plate 3.17(A•I) Lip (Character 49) of (A) S. gracilis, (B) Spathoglottis 103 cf. vanvuurenii, (C) S. kimballiana var. kimballiana, (D) S. hardingiana, and (E) S. pubescens. Callus (Character 66) of (F) S. plicata, (G) S. affinis, and (H) S. aurea. Auricle/teeth (Character 64) of (I) S. aurea. A

Plate 3.18(A•H) Column (Character 70); side view of (A) S. 104 microchilina, (B) S. aurea, (C) S. unguiculata, (D) S. pubescens, (E) S. hardingiana, (F) S. plicata; dorsal view, (G) S. lobbii, and (H) S. affinis.

Plate 3.19(A•F) (A) Pollen of S. affinis, (B) pollen of S. plicata, (C) 105 pollinarium of S. plicata var. alba, (D) pollen of S. microchilina, (E) pollinarium of S. kimballiana var. kimballiana, and (F) pollen of S. kimballiana var. kimballiana; caudicles removed.

C Plate 5.1 Early illustrations on the flower and labellum of S. 194 aurea by Johannes Jacobs Smith in his Die Orchideen von Java (1909).

Plate 5.2(A•H) S. aurea (A) habit, (C) flower, (F) inflorescence, (G) 196 self•pollinated flowers and fruits; and S. microchilina (B) habit, (D) flower, (E) inflorescence, (H) self•pollinated flowers and fruits. (Pictures not following scale).

Plate 5.3(A•H) Diverse forms of Spathoglottis aurea flowers. (A-D) 208 insect-pollinated flower, (E-F) geitonogamous flowers, and (G-H) cleistogamous buds with swollen ovary. (Pictures do not following scale).

Plate 5.4 Variations in the shape of the labellum/lip of 209 Spathoglottis aurea.

Plate 5.5 Variations in the shape of the labellum/lip of 210 Spathoglottis microchilina.

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LIST OF ABBREVIATIONS

asl above sea level bp base pairs c. approximately cf. confer to cm centimeter DNA Deoxyribonucleic Acid dNTP Dinucleotide Triphosphate E East e.g. example et al. And others (et alia) GPS Global Positioning System km² kilometer square Kya thousand years ago L Liter lat. latitude long. longitude m meter M Molar mM millimolar min minutes ml milliliter mm millimeter Mya million years ago N North no. number PCR Polymerase Chain Reaction pers. comm. personal communication rpm revolutions per minute sec seconds sp. species (singular) spp. species (plural) V Volt v/v volume over volume var. variety Ver. Version w/v weight over volume ~ approximately < less than > more than ≤ similar and less than ≥ similar and more than • to

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% percent & and / or µ micro °C degree Celcius

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KAJIAN FILOGENETIK GENUS Spathoglottis Blume (ORCHIDACEAE) DI

MALESIA

ABSTRAK

Genus Spathoglottis Blume (tribe Collabieae, subfamili Epidendroideae) adalah genus dengan sejumlah 49 spesies geofit tanah dengan taburan di seluruh tropika dan subtropika Asia sehingga ke Kepulauan Pasifik. Sejumlah 44 spesies telah direkodkan di rantau Malesia dan tertumpu di New Guinea. Walaupun terkenal dari segi hortikultur,

Spathoglottis telah dikenalpasti sebagai satu genus dengan kecelaruan taksonomi di dalam famili Orchidaceae. Sebagai contoh, kecelaruan telah dikenalpasti di antara dua spesies Spathoglottis yang berbunga kuning; iaitu S. aurea dan S. microchilina yang mana kedua-dua spesies menunjukkan keplastikan (kepalsuan) morfologi yang kompleks. Oleh yang demikian, kajian ini telah dimulakan untuk mengenalpasti ciri-ciri morfologi di antara spesies Spathoglottis, seterusnya kajian filogenetik molekul dijalankan bagi menjelaskan hubungan di antara spesies di dalam genus ini. Sejumlah 16 taksa (13 spesies dan tiga varieti) Spathoglottis dari Indo-Cina dan seluruh Malesia telah diperoleh. Sebanyak 72 ciri-ciri morfologi dan tiga ciri ekologi telah dikenalpasti dari semua taksa yang dikaji. Berdasarkan analisis dari segi morfologi, genus Spathoglottis di

Malesia telah terbahagi kepada dua pengkelasan utama berdasarkan warna bunga iaitu:

(1) Kompleks Spathoglottis Ungu dan (2) Kompleks Spathoglottis Kuning. Pengkajian molekul untuk spesies Spathoglottis telah dijalankan dengan menggunakan kaedah penanda molekul dari dua plastid DNA gen (matK dan trnL-F) dan internal transcribed spacer dari ribosoma nuklear DNA (ITS). Tiga analisis filogenetik telah dijalankan untuk mengkaji hubungan evolusi di antara spesies Spathoglottis di Malesia iaitu:

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Maximum Parsimony (MP), Maximum Likelihood (ML) dan Bayesian Inference (BI).

Analisis gabungan untuk data urutan nukleotida dari penanda molekul plastid dan nuklear telah menunjukkan bahawa genus Spathoglottis adalah monofiletik dengan indeks konsistensi, CI= 0.709; indeks pengekalan, RI= 0.906; peratusan bootstrap, BS=

98% (ML), BS= 100% (MP) dan nilai kebarangkalian posterior, PP= 1.0 (BI). Hasil dari analisis ini, empat kumpulan utama telah dikenalpasti iaitu: (1) Dwarf Spathoglottis

Ungu, (2) Dwarf Spathoglottis Kuning, (3) Large Spathoglottis Ungu, dan (4) Large

Spathoglottis Kuning. Perpecahan di antara kumpulan Dwarf dan Large Spathoglottis menunjukkan telah terjadinya perbezaan awal dari segi saiz tumbuhan, warna bunga, dan saiz bunga; berkemungkinan dari perubahan strategi persenyawaan kasmogami

(penyebaran/persenyawaan oleh serangga/luar) kepada kleistogami (persenyawaan sendiri/dalaman). Berdasarkan analisis molekul tersebut, satu varieti Spathoglottis yang berbunga putih, S. plicata var. alba telah dicadangkan untuk diubah dari segi penamaan taksonominya, dan dinaik taraf dari status varieti kepada spesies; manakala identiti satu spesies Spathoglottis dari Borneo, iaitu S. kimballiana turut memerlukan kajian lanjut.

Walau bagaimanapun, pohon morfologi yang diperoleh dari kajian ini didapati tidak kongruen dengan pohon-pohon dari analisis molekul, membuktikan bahawa sekiranya hanya dengan menggunakan data morfologi, keputusan yang diperoleh boleh mewujudkan kekeliruan kerana ciri-ciri morfologi bunga berkeupayaan untuk berubah- ubah. Keputusan analisis morfologi, molekul, dan jurang kod bar DNA menunjukkan bahawa S. aurea dan S. microchilina sebagai dua spesies yang berbeza. Keputusan dari analisis-analisis ini telah digunakan untuk mengkelaskan spesies kepada taburan kepulauan geografi yang tertentu. Didapati, setiap spesies Spathoglottis hanya boleh dijumpai di kepulauan tertentu di dalam Malesia; dengan hampir tiada spesies

xxxi menyeberangi kepulauan yang berbeza; kecuali spesies yang paling luas taburannya iaitu

S. plicata. Oleh yang demikian, lima kepulauan yang merupakan habitat asal kepada spesies Spathoglottis tersebut telah dikenal pasti di dalam kajian ini iaitu: (1) Indo•Cina,

(2) Semenanjung Malaysia + Sumatra (Malesia Barat), (3) Borneo (Malesia Barat), (4)

New Guinea + New Caledonia (Malesia Timur) dan (5) Meluas (Malesia Timur +

Malesia Barat).

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PHYLOGENETIC STUDY OF GENUS Spathoglottis Blume (ORCHIDACEAE)

IN MALESIA

ABSTRACT

The genus Spathoglottis Blume (tribe Collabieae, subfamily Epidendroideae) is a genus with a total of 49 terrestrial geophyte species and is widely distributed in tropical and subtropical Asia and the Pacific Islands. A total of 44 species were recorded in the

Malesian region and are concentrated particularly in New Guinea. Despite of being popular in horticulture, Spathoglottis is a taxonomically confused genus in Orchidaceae.

For example, confusion is detected between a pair of species within the yellow-flowered

Spathoglottis; the S. aurea and S. microchilina with both species show a complex morphological plasticity. This study was initiated to examine the morphological attributes among species of Spathoglottis; and establishing a molecular phylogenetic study in elucidating the relationships among members of the genus. A total of 16 taxa (13 species and three varieties) of Spathoglottis from Indo•China and throughout Malesia were examined. Seventy•two morphological and three ecological characters were selected from all taxa under investigation. Based on the morphological analysis, genus

Spathoglottis in Malesia can be divided into two major sections based on the colour of the flower: (1) the Purple Spathoglottis Complex and (2) the Yellow Spathoglottis Complex.

Molecular studies for the species of Spathoglottis were carried out using two plastid DNA genes (matK and trnL•F) and the internal transcribed spacer of a nuclear ribosomal DNA

(ITS). Three phylogenetic analyses were conducted to study the evolutionary relationships among species of Spathoglottis in Malesia: Maximum Parsimony (MP), Maximum

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Likelihood (ML) and Bayesian Inference analyses (BI). The analyses of combined plastid and nuclear data showed that genus Spathoglottis is monophyletic with consistency index,

CI= 0.709; retention index, RI= 0.906; bootstrap percentages, BS= 98% (ML), BS= 100%

(MP) and posterior probability value, PP= 1.0 (BI). Four major groups were determined from these analyses: (1) Dwarf Purple Spathoglottis, (2) Dwarf Yellow Spathoglottis, (3)

Large Purple Spathoglottis, and (4) Large Yellow Spathoglottis. The split in the Dwarf and Large Spathoglottis Groups might reflect an early differentiation of plant size, flower colour, and flower size; perhaps due to the shift from chasmogamy (insect-pollinated) to cleistogamy (self-pollination) strategies. From these analyses the white-flower

Spathoglottis, S. plicata var. alba is proposed for taxonomical and nomenclatural changes by upgrading it to a species rank; whereas the identity of a Bornean species, S. kimballiana needs further investigation. However, the morphology tree obtained in this study was incongruent to the molecular trees and morphology alone can be misleading for inferring the relationships among groups of interest, as floral morphology is highly flexible. The morphological, molecular, and DNA barcoding gap analyses showed S. aurea and S. microchilina as two separate species. These results were used for grouping of species into their specific geographical island distributions. Certain species of Spathoglottis were observed to confine to only particular island groups throughout Malesia; with almost a complete no crossing-over between the islands; except for the most widespread and weedy species, S. plicata. Thus, five island groups from the West, Central and East Malesia were proposed in this study: (1) Indo•China, (2) Malay Peninsula + Sumatra (West Malesia),

(3) Borneo (West Malesia), (4) New Guinea + New Caledonia (East Malesia) and (5)

Widespread (East Malesia + West Malesia).

xxxiv

CHAPTER ONE

INTRODUCTION

1.1 General Introduction

The orchid family, Orchidaceae is one of world’s two largest families of flowering plants. It is also richly diverse, with over 25,000 named species in 736 recognized genera, and perhaps second only to the sunflower family, Asteraceae (Compositae).

Orchidaceae comprises a substantial ten percent of all worlds’ vascular plant species, with at a rate of roughly 500 new species and 13 new genera being described every year

(Dressler, 1993; Freudenstein and Rasmussen, 1999; Chase, 2005; Chase et al., 2015).

The orchid family is highly evolved and their diversity of specialized individual pollinators or mycorrhizal fungi, adaptive ecological and pollination strategies, evolution of pollinia and rapid degree of speciation have provided a rich system to study on evolutionary patterns (Freudenstein and Rasmussen, 1999; Givnish et al., 2015).

Traditionally, the classification systems of the orchids were based on subjective assessments of the appearance of the whole plant and their morphological characters

(Dressler, 1981; Dressler, 1993; Freudenstein and Chase, 2015). Since the past two centuries, the orchid systematics have been based exclusively on features of the flower, predominantly the gymnostemium or column and anther; which are unique to this family

(Freudenstein et al., 2002; Chase et al., 2003; Xiang et al., 2014). However, classification that relies heavily on characters was not warranted as their structure is purely intuitive. They often show considerable convergence due to ecological selection

1 and create problem at higher levels because floral morphologies are extremely plastic

(Chase, 2005; Miner et al., 2005; Górniak et al., 2010; Ackerman et al., 2011; Paniagua-

Ibáñez et al., 2015).

The past 20 years have shown significant progress in the systematics of orchids when molecular data have come to play an important role in angiosperm classification.

Numerous DNA phylogenetic studies have shed new lights into the relationship of the orchid family. Increasing efforts are being focused on familial•level classification; ranging from genera (Bellstedt et al., 2001; Jheng et al., 2012; Xiang et al., 2013) to subtribes (Cameron, 2005; Sosa, 2007) to tribes (Van Den Berg et al., 2005; Xiang et al.,

2014) to subfamilies (Freudenstein et al., 2002; Kocyan et al., 2004; Freudenstein and

Chase, 2015) and to the whole family (Chase et al., 2003; Chase, 2005; Chase et al.,

2015). Following the most recent classification system of Orchidaceae based on molecular data (Chase et al., 2015) the family of orchids were recognized into five subfamilies; the Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae and

Epidendroideae.

The genus Spathoglottis Blume (tribe Collabieae, subfamily Epidendroideae) is a well- known genus with a total of 49 terrestrial geophyte species and is widely distributed in tropical and subtropical Asia and the Pacific Islands; where 44 species were recorded in the Malesian region and are concentrated particularly in New Guinea (Govaerts, 2013).

Blume proposed the genus Spathoglottis in 1825 with S. plicata as the type species and the only known species at the time. In 1838, Lindley described the genus as Paxtonia

2 which is now regarded as a synonym of Spathoglottis. According to Cribb and Tang

(1981), despite of being popular in horticulture, Spathoglottis is a taxonomically confused genus in Orchidaceae. From the tribal level itself, placement of tribe

Collabieae (subfamily Epidendroideae) has been hotly debated over the years.

Collabieae has not been recognized by most authors and was considered as synonym to

Bletiinae (Sosa, 2007; Xiang et al., 2014). Dressler (1993) treated it as groups of uncertain systematic position thus listed it as one of his “leftover and misfits” groups.

Collabiinae was an unplaced subtribe in previous classifications but is now elevated to a tribe based on the most recent revision done by Chase et al. (2015).

Early revision work on this genus was discussed by Dockrill (1969) for the Australian

Spathoglottis species, followed by Cribb and Tang (1981) for Australia and the Pacific

Islands whilst Hallé (1977) revised the New Caledonian species. However, a revision of

Spathoglottis in Malesia is still lacking until now and no molecular phylogenetic study has been carried out. Studies of the morphology and the anatomy have been reported by several authors such as in Holttum (1964) and Seidenfaden and Wood (1992).

Observations on the leaf anatomy of Spathoglottis were made by Solereder and Meyer in

1930 and Williams in 1979; and on the root by Pridgeon et al. in 1983 and later by

Porembski and Barthlott in 1988. Teoh (1984) and Brandham in 1999 reviewed the cytogenetics part and suggested dysploidy in Spathoglottis (chromosome count of

2n=18, 38, 40, 42, and 60). To date, no palynological study of Spathoglottis has been published, although pollen characters were suggested to be taxonomically informative.

Recently, a molecular study on the chloroplast DNA barcoding of Spathoglottis in

Malaysia for genetic conservation has been carried out by Ginibun et al. (2010) and four

3 chloroplast regions, matK, rbcL•a, rpoB and rpoC1 were successfully amplified from all seven species (S. aurea, S. gracilis, S. kimballiana, S. plicata, S. plicata var. alba, S. unguiculata and a Spathoglottis hybrid) treated. However, their DNA barcoding study does not provide a deep phylogenetic inference, as the main goal is not to determine patterns of relationship but to identify an unknown sample in terms of a preexisting

Spathoglottis classification.

Morphologically, confusion is detected between a pair of species within the yellow- flowered Spathoglottis; the S. aurea and S. microchilina complex. This observation was also reported by Seidenfaden and Smitinand (1959), Wood (1997), Chan et al. (2001),

Comber (2001) and Seidenfaden and Wood (1992). The two species in this complex is distinguished based on the width of the lip (1.5 mm in S. microchilina; 4 mm wide in S. aurea) and the ability of the flower of S. microchilina to self-pollinate (cleistogamy). In the wild, populations of both S. aurea and S. microchilina show great phenotypic variations and forms. Thus, further examination is required to delimit S. aurea and S. microchilina as two distinct species or just one highly variable form of S. aurea.

On a biogeographical note, it is very informative to look at the distribution patterns among the species of this genus. Throughout Malesia, certain species of Spathoglottis were observed to confine to only particular island groups; with almost a complete no crossing-over between the islands; except for the most widespread and weedy species, S. plicata. The seven island groups observed were: Indo-China, Malay Peninsula +

Sumatra (Sunda Shelf), Borneo (Sunda Shelf), Java (Wallacea), Sulawesi (Wallacea),

4 the Philippines (Wallacea) and New Guinea (Sahul Shelf). Vicariance hypothesize was assumed for the dispersal pattern of Spathoglottis through Malesia and intercontinental long-dispersal is relatively uncommon or impossible. This is suggested by comparing the age of the Orchidaceae that have arisen roughly 112 Mya ago (Late Jurassic to

Cenozoic) and the most recent rapid divergence in higher epidendroids (subfamily

Epidendroideae, tribe Collabieae) was estimated between 30.8–37.9 Mya (Christenhusz and Chase, 2013; Givnish et al., 2015). The West Malesia islands (Sunda Shelf) were already in place approximately 160 Mya, and the East Malesia islands (Sahul Shelf) amalgamated at ~50 Mya and most of the Wallacea (Sulawesi, the Philippines and

Lesser Sunda Islands) has only emerged at about 5 Mya (Cenozoic); providing evidence that there was no land bridge possible but might only be stepping stones (Hall, 1999;

Voris, 2000; Van Welzen and Slik, 2009; Van Welzen et al., 2011).

1.2 Objectives

Phylogenetic study among species within Spathoglottis has never been established. This is a pressing need since establishing species relationships will address issues such as evolutionary relationship between species as well as determination of identity or taxonomic status of certain species.

Thus, the objectives of this study are:

1) To examine the morphological characters among species in genus Spathoglottis

and selecting key characters as important features in identification and placement of

5

taxa

2) To assess how useful a plastid gene (matK), non-coding plastid marker (trnL•F), the

internal transcribed spacer (ITS) of the nuclear ribosomal DNA and combined

molecular sequences in elucidating species relationship among members of

Spathoglottis; and analyze their correlation to the morphological data

3) To investigate the biogeographical pattern among species in genus Spathoglottis

based on the internal phylogeny groupings

4) To resolve the taxonomic questions in Yellow Spathoglottis Complex of S. aurea

and S. microchilina

6

CHAPTER TWO

LITERATURE REVIEW

2.1 What and Where is Malesia?

The Malay Archipelago, also known as Malesia is one of the three world’s richest tropical rainforests with estimated 42,000 spp. (Van Welzen et al., 2005). Malesia is a plant geographical region that what first recognized and described by Heinrich

Zollinger, a Swiss botanist and explorer in 1857. In his article, Zollinger noticed that the flora of this huge archipelago was in many respects, very distinct from that of neighbouring regions (Raes and Van Welzen, 2009; Schuiteman, 2013). He named his floristic region as ‘Flora Malesiana’, after the Malay language that is commonly used throughout the archipelago.

Malesia was defined based on its flora distribution patterns, and the recognition on the presence or absence of species throughout the archipelago. Zollinger strictly acknowledged that a floristic region should never be interchanged with the boundaries of political colonies. Relying on a very limited plant distribution data; and coupled with many straight lines; in 1857 Zollinger defined the boundaries of the floristic region of

Malesia (Figure 2.1; total grey area). Many of his colleagues at the time argued that his delimitation is too extensive. Responding to it, Zollinger thus recognized ‘Flora

Malesiana’ in a more restricted sense (dark•grey area). However, Zollinger largely

7

Figure 2.1: Map of the boundaries in Malesian floristic region as recognized by Zollinger in 1857. Total grey area is in the widest sense and the dark•grey area in more restricted sense. New Guinea is largely excluded. The delimitation by Van Steenis (1948; 1950) is marked by three demarcation knots (red circle). The numbers indicate the number of plant genera not crossing the knots. The different lines indicate Wallace’s Line and its variants by different authors that split up Malesia to the eastern and western parts. Map adopted from Raes and Van Welzen (2009).

8 excluded New Guinea from his Malesian flora due to lack of collections. He also sighted snows on the highland’s peak of New Guinea mountains; which led him to conclude that flora of New Guinea much resembled those in temperate mainland than of an island flora (Van Welzen et al., 2005; Raes and Van Welzen, 2009).

Ever since the recognition of Malesia, debates have been going on about the internal division of this floristic region. One of the world’s most prestigious flora projects, the

‘Flora Malesiana’ was first thought to be defined by Van Steenis (1948). He indeed circumscribed the area; however the term is older and was introduced earlier by

Zollinger in 1857. Only few people were aware of this and most of the time they have been referring to incorrect references; duly to the fact that Zollinger’s publication was written in Dutch therefore unreadable for most of the non•native.

In 1948, Van Steenis came with the idea to develop a Flora of Indonesia; which then was still Dutch•Indonesia. However, he did not plan to produce a national flora, but instead he wanted to compile a flora based on a phytogeographical region; an area with many endemic species and elements of its own (Van Welzen et al., 2005). Despite of most of the plant species were still poorly known at that time, Van Steenis (1948; 1950) managed to identify three sharp boundaries based on the distribution of 2178 plant genera with shared geographical limit: (1) the most Western boundary (between

Thai•Malaysian borders) where 375 genera have northern limit and 200 genera reached

9 their southern limit, (2) the most Northern boundary (between the Philippines•Taiwan) with 421 genera to the south and 265 genera to the north of it, and (3) the most Southern boundary (between New Guinea and Australia) with 340 genera to the south and 644 genera to the north of it, respectively (Figure 2.1). Almost after 100 years later, Van

Steenis largely confirmed Zollinger’s initial delimitation of Malesia floristic boundaries.

2.1.1 Wallace’s Line – Two or Three Phytogeographical Areas?

Wallace’s Line or its variants (Figure 2.1) were known to divide Malesia into a western

(Sunda Shelf) and eastern sub•region (Sahul Shelf).

Throughout his nine years of expedition to South East Asia, Alfred Russell Wallace

(1860) noticed that the fauna of the Malay Archipelago consisted of Asian and New

Guinea•Australian elements; which then led him to the discovery of a famous zoological boundary of all time. The invisible line, known as Wallace’s Line runs east of the Philippines, between Borneo and Sulawesi and finally between Bali and Lombok.

Wallace documented his finding in series of scientific articles and books; and receiving crowds of attention through the award•winning The Malay Archipelago (Wallace,

1890). He observed that the distributions of animal groups within the archipelago discontinuous across the line. He however was uncertain on the position of Sulawesi and called it as ‘an anomalous island’ with old endemic Australian species and lacks continental Sundaic groups (Turner et al., 2001; Raes and Van Welzen, 2009; Van

Welzen et al., 2011; Crayn et al., 2015). The other authors have recognized similar lines;

10 the western and eastern variants of Wallace’s Line (Merill­Dickerson/Huxley Line and

Zollinger’s Line) that split up Malesia into its western and eastern parts. Wallace’s Line and its variants separated Malesia into two major areas based on its fauna composition; but are they suitable to explain the plant distribution patterns in this region?

Numerous studies focusing on the composition of plant families and genera in Malesia; as prepared by Baker et al. (1998); Cox (2001); Van Welzen et al. (2005), Raes and Van

Welzen (2009), Van Welzen and Slik (2009); Van Welzen et al. (2011) and Webb and

Ree (2012) have finally revealed a stronger partitioning of Malesia into three instead of two phytogeographical areas: the western Sunda Shelf, central Wallacea and eastern

Sahul Shelf. This will be further discussed in subtopic 2.3.

2.1.2 The Demarcation and Internal Division of Malesia

The status of Malesia as a phytogeographical region was recently confirmed by the studies on plant distribution patterns and plate tectonics in Malesia.

Malesia reaches from the southern tip of Thailand, stretched throughout Malaysia and

Indonesia to the Philippines and Papua New Guinea (Figure 2.2). It is comprises of three higher phytogeographical subunits: (1) the western Sunda Shelf (2) central Wallacea, and (3) the eastern Sahul Shelf which includes nine island groups: Malay Peninsula (not a true island), Sumatra, Borneo, Java, Sulawesi, the Philippines, Moluccas, Lesser Sunda

Islands and New Guinea.

11

Legend

Malesia

Figure 2.2: Map of Malesia. Re-draw from Van Welzen et al. (2011).

12

The western part of Malesia is the everwet Sunda Shelf comprising of the Malay

Peninsula, Sumatra and Borneo (minus Java) (Figure 2.3). The central Malesia known as

Wallacea is a transition zone between the flora of Sunda and Sahul. Wallaceae has a dry monsoon climate throughout the year; and Sulawesi, Moluccas, the Philippines

(including Palawan and Mintoro), Lesser Sunda Islands and Java were islands grouped together in Wallacea. Java was previously placed under Western Malesia. However, Van

Welzen et al. (2011) reported that Java shares Wallacean flora and has a dry monsoon climate. Wallaceae is a distinct floristic area as it comprised many endemic and drought•tolerant elements. New Guinea (Indonesian Irian Jaya and Papua New Guinea) made up the Sahul Shelf.

As parallel to Van Steenis (1948; 1950), three invisible ‘demarcation knots’ or clear borders based on generic distributions surrounding Malesia were recognized: (1) between the southern tip of Thailand throughout Malaysia and Indonesia, (2) between

Taiwan and the Philippines, (3) between New Guinea and Australia. In either direction, these borders are not crossed by numerous plant genera and species; which the orchids

(Orchidaceae), Nepenthes (Nepenthaceae), Lithocarpus (Fagaceae) and Ericaceae provide many examples of these (Van Welzen et al., 2005; Raes and Van Welzen, 2009;

Van Welzen and Slik, 2009; Schuiteman, 2013).

13

Figure 2.3: Three phytogeographical subunits in Malesia: Sunda, Sahul and Wallacea. The area of endemism employed and intercontinental dispersal dynamics. Map adopted from Crayn et al. (2015).

2.2 Plate Tectonics and Changing Palaeogeography in Malesia

Malesia is a mosaic of colliding smaller and major plates with many small tectonic fragments or slivers acting as ball•bearings. During the ancient tectonic movements, several seas meets; the islands arcs collided with continents and continental fragments; leading to the rise of mountains and formation of deep ocean basins. East, south and west of Malesia were surrounded by volcanic arcs belt with abundant volcanism and intense seismicity; due to the high lithosphere subductions of the Indian and Pacific

14

Oceans. The geology and palaeogeography of Malesia continues to change rapidly and the region has characterized most of the Cenozoic (~0•66 Mya) (Voris, 2000; Van

Welzen et al., 2005; Hall 2009).

The Eurasian Plate moves slowly to the east due to the opening of the Atlantic Sea while the Pacific Plate is moving west and disappears below the Eurasian and New

Guinean•Australia Plate. During its movement towards north, many tectonic fragments and slivers broke off from the New Guinean•Australia Plate. Meanwhile, the Indian

Ocean Plate moves to the east and subducting below the Malay Archipelago belt

(Sumatra to Lesser Sunda Islands). Finally, the Philippine Plate has almost disappeared and left only with some continental debris (Van Welzen et al., 2011).

The broke off tectonic slivers from the major continental plates can be simply overviewed in two waves (Audley•Charles, 1987). The first wave constitutes of the present day South East Asia mainland (Burma, Tibet) and West Malesia (Sunda Shelf) that includes the Malay Peninsula, Borneo, Sumatra and Java. This wave broke off from

Australia and collided with the Eurasian Plate while rafting northwards. The process was estimated to start at least during the Late Jurassic (~160 Mya) (Van Welzen et al., 2011) but it may have been earlier (Palaeozoic, ~400 Mya) (Voris. 2000; Turner et al., 2001;

Hall, 2009). Importantly to note, these areas already formed the present South East Asia

(although of Australian origin) long before the evolution of many recent plants and animals. Most of plant families existed as early as the late Cretaceous (~70 Mya) and relatively earlier (~120 Mya) for the orchids (Christenhusz and Chase, 2013). Thus, the

15 present-day flora and fauna will be mainly of South East Asia origin (Cox and Moore,

1993; Turner et al., 2001; Van Welzen et al., 2011).

The second wave broke off much later, approximately 50 Mya and formed the East

Malesia (Sunda Shelf • Sulawesi, Moluccas, Lesser Sunda Islands and New Guinea).

Most of the East Malesia areas; certainly, New Guinea, Sulawesi and the Philippines were amalgamations of microplates. The Sunda Shelf and Sahul Shelf were widely separated by ocean. They were only brought into contact ~25 Mya (early Miocene) during the northwards transition of Sahul after breaking off from Antarctica. The collision has caused extensive uplift and orogenesis events in the archipelago and emergence of numerous islands in Central Malesia; known today as the Wallaceae. Even when the islands in the Central Malesia reached their present-day placements, most areas were still submerged during the time and only started to emerge during the Pliocene approximately 5 Mya (Pigram and Davies, 1987; Van Welzen et al., 2005; Hall, 2009).

As for New Guinea, it was a complicated history. Originally, New Guinea was only consisted of the southern part termed ‘Craton’ which has always been attached to

Australia. The rest of New Guinea was amalgamations of more than 30 tectonic slivers or terranes of various origins (island arcs, continental fragments and pieces of sea floor) that formed the present-day Peninsula in the east and northern coast; and the Bird’s head in the west (Pigram and Davies, 1987; Van Welzen et al., 2005; Hall, 2009; Van Welzen et al., 2011).

16

For the Philippines, only parts of Luzon were left as continental debris from the original

Philippine Plate. Borneo, Palawan and Mintoro were slivers that broke off from the mainland South East Asia. The rest of the Philippines arrived with the tectonic slivers of the New Guinean•Australian plate during the second wave (Van Welzen et al., 2005).

Thus, the final stepping stones between South East Asia mainland and Australia got into position with this second wave.

2.3 Plants Distribution Patterns and Floristic Exchange in Malesia

Tectonic movements and rapid orogeny events in Malay Archipelago have opened up new niches and dispersal opportunities for its flora and fauna; predominantly in the

Pliocene (~5.3•2.6 Mya). Climatic fluctuations accompanied by lowering and rising of sea levels during the late Pleistocene (~12 Kya•250 Kya); land bridge systems and sea barriers together with the uplift of highlands in Sumatra, Java, Sulawesi and New

Guinea have promotes niches diversity throughout this region (Voris, 2000; Van Welzen et al., 2005; Hall, 2009; Crayn, 2015). The radiation of species and rapid speciation events in Malesia were hypothesized as either through long dispersal of taxa or vicariance (disjunction) due to habitat fragmentations (Thomas et al. 2012; Christenhusz and Chase, 2013). The origins of the Asian and Australian species; and migrations between them in Malesia have long fascinated biologists. Many flora and fauna were observed to have a discontinuous distribution across the Wallace’s Line; which confine extant biotic groups to their component areas (Figure 2.3).

17

Malesia is a natural phytogeographic area with about 70% of its total flora is endemic to this region. Among other richest plant families with most endemic species are Ericaceae,

Moraceae, Dipterocarpaceae and the orchids (Orchidaceae; with roughly 6800 species known to Malesia and 88% are endemics) (Van Welzen and Slik, 2009; Schuiteman,

2013).

The flora richness of each island groups in Malesia: Malay Peninsula, Borneo, Sumatra,

Java, Sulawesi, the Philippines, Moluccas, Lesser Sunda Islands and New Guinea were observed correlates significantly with the size of the island subunits. All the nine areas show high species endemism with Borneo, the Philippines and especially New Guinea comprise significantly highest number of endemic species (Turner et al., 2001; Van

Welzen and Slik, 2009; Van Welzen, 2011; Crayn, 2015).

In 2005, Van Welzen et al. reviewed the plant distribution and composition along the

Wallace’s Line. Their treatments were subsequently followed by works of Raes and Van

Welzen (2009) and Van Welzen et al. (2011). They analyzed the distribution patterns among 7340 species of plants from 165 families and 896 genera; which represents about

25% of total Angiosperms and ferns in Malesia. Botanical evidence from their studies revealed a stronger partitioning of Malesia into three phytogeographical areas instead of two: Sunda, Sahul and Wallaceae. Differences in climate explained the groupings of flora in the tree subregions which Sunda and Sahul Shelves are everwet whereas

Wallaceae experienced dry monsoon climate throughout the years.

18

During the Glacial Maxima (Cenozoic); Sunda and Sahul Shelves formed landmass connected with Asia and Australia thus dispersal and exchange of species on the areas were relatively easy; whereas sea barriers remained within Wallacea. There was no major land bridge present in Malesia during the glacial periods and most stepping stones for species dispersal were only emerged in the last ~10 Mya (Hall, 1999; Voris 2000;

Van Welzen at al., 2011).

Consequently, the species composition of the two shelves is more homogenous as compared to Wallacea which consisted of many drought•tolerant species. It is also apparent that whatever line is used (Wallace’s Line and its variants); they are all good boundaries and moving west to east shows stronger demarcation line. Lowest amount of species is crossing the Lydekker’s Line which separates New Guinea from the rest; and this can be explained by the sea barriers surrounding Wallacea predominantly the deep

Makassar Straits (Voris, 2000).

Similar studies on biogeography inference at generic and family level in Malesia were undertaken by Baker et al. (1998); Turner et al. (2001); Van Welzen et al. (2005);

Brown et al. (2006); Raes and Van Welzen (2009); Van Welzen and Slik (2009);

Schuiteman (2013); Crayn et al. (2015); and also involving the use of molecular markers to infer evolutionary relationships among the taxonomic units in Malesia as in Wagstaff

(2004); Gussarova et al. (2008); Micheneau et al. (2008); Knopf et al. (2011); Thomas et al. (2012) and Christenhusz and Chase (2013). They observed the same patterns in

19 plant distribution and groupings along the Wallace’s Line thus supporting Van Welzen et al. (2011).

2.4 The Orchids Family

The Orchidaceae is one of the largest families in flowering plant kingdom and one of the most actively evolves, diverse and widespread. To date, over 25,000 species of orchids in 736 genera were recognized; placing them probably second to the sunflower family,

Asteraceae (Compositae). A substantial ten percent of all worlds’ vascular plant species are the orchids; with at a rate of roughly 500 new species and 13 new genera being described every year. New knowledge from molecular studies has classified orchids into five subfamilies; the Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and

Epidendroideae (Dressler, 1993; Freudenstein and Rasmussen, 1999; Pridgeon et al.,

1999; Chase, 2005; Pridgeon et al., 2005; Chase et al., 2015).

Orchids are cosmopolitan and among the most well•adapted plants. They strive on wide range of habitat worldwide; distributed on all vegetated continents except for the driest and coldest region; the deserts and Antarctica. The orchids are however most abundant in the humid tropics and subtropics where their true homes are.

Eventhough they often thought as rare; orchids in a sense are quite commonplace. They can be found anywhere; dwelling on the wet and shaded forest floor among leaves litters of the lowland tropical rainforest, on the branches of tall forest trees exposed to direct

20 sunlight and heavy rainfall for hours, in grassy and swampy areas of landslips and bogs, among rocky crevices and mossy ridges near the summit of a mountain, pioneering in left–over opening and by the roadside ditches or waterfalls, riversides or sea shore, and even on the margin of a forest (Dressler, 1981; Dressler, 1993; Pridgeon et al., 1999;

Ong et al., 2011).

As their wide distribution is concerned, Orchidaceae has inspired a great deal of speculations on their timing and place of evolution. Fossil evidence of the orchids from the Meiocene has strongly suggested Orchidaceae as an ancient group which their origin predates the break•up of the Gondwanaland 125 Mya (Pridgeon et al., 2001). A recent time•calibrated phylogeny study by Givnish et al. (2015) has estimated that

Orchidaceae have arisen roughly 112 Mya during the Late Jurassic. Subfamily

Epidendroideae represents the pinnacle of orchids’ evolution (~37.9•30.8 Mya) which supported by rapid divergence among taxa in the subfamily (Givnish et al., 2015; Chase et al., 2015).

2.4.1 Diversification of the Orchidaceae

Variability in plant vegetative structures and size, pollen evolution, habitat adaptation, specific individual mycorrhizal fungi, and specialized pollination strategies have contributed to the significant diversification of Orchidaceae (Dressler 1993;

Freudenstein and Chase, 2015; Givnish et al., 2015).

21

The orchids can be epiphytic which grow high up on tree boles and branches; the ground•dwellers (terrestrial/heteromycotrophic), or lithophytic that grows among rocky boulders and limestone crevices (Plate 2.1A•F).

Majority of the orchids are epiphytic which constitutes nearly 80% of total species count and found mostly in tropical forests at montane altitude of 1000•1500 m asl; whereas all temperate orchids are of terrestrial species (Dressler, 1981; Atwood, 1986; Seidenfaden and Wood 1992; Rasmussen, 1995; Pridgeon et al., 1999; Krömer et al., 2005).

Epiphytism among else has been suggested to accelerate both speciation and extinction rates in Orchidaceae (Givnish et al., 2015). Diverse vegetative structures and thousands of tiny seeds that capable for long•distance dispersal are among key innovations in epiphytic orchids. Spongy velamen root, waxy leaves, and fleshy pseudobulb were developed in response to adaptation on various adverse environmental needs; especially the problems of temperature fluctuation and water conservation on daily and seasonal basis which is very crucial on the tree canopies (Dressler, 1981; Pridgeon et al., 1999).

The tiny seeds, brought along by the wind will eventually settled down on the tree branches; invaded new niches that largely unoccupied by other vascular plant (Atwood,

1986).

22

A B

C D

E F

Plate 2.1(A•F): Epiphytic orchids (A) Agrostophyllum majus, (B) Grammatophyllum speciosum, (C) Dendrobium hasseltti; lithophytic orchid (D) Ludisia discolor; and terrestrial orchids (E) Anoectochilus geniculatus, (F) Corybas holttumii in their natural habitat. (Pictures not following scale).

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As epiphytism is often associated with tropical montane condition; deep valleys and high ridges of the mountains may provide physical barriers to gene flow due to the isolation of populations at larger spatial scale. While concurrently, the tiny seeds through their long•distance dispersal permitting parallel genetic differentiation in many sites along the extensive mountain area. These together will further accelerate both speciation and extinction rates in epiphytic orchids. However, it is relatively opposite in their terrestrial counterparts (Givnish et al., 2015).

Due to limited food reserves in the endosperm; all orchids predominantly terrestrial species rely heavily on specific mycorrhizal fungi for germinating and growing; either partly (heteromycotrophic) or remains obligate for life (holomycotrophic/saprophytic)

(Arditti, 1967; Ramsay et al., 1986; Whigham and Wilhems, 2003; Swarts, 2009). These fungi will provide resources for germination and carbon capture during their protocorm stage. When moving terrestrial orchids to different areas in absence of their specific individual mycorrhizal fungi; germination will unlikely to happen. This orchid•mychorrizal relationship paired with their habitat preferences explained the lower speciation rate in the terrestrial groups (Rasmussen, 1995; Givnish et al., 2015).

2.5 The Orchid Flower

Traditionally in Orchidaceae, the identification of species and delimitation of taxa were weighted heavily on morphology; both vegetative and primely the flower. The features of their anthers (erect, reflexed or incumbent), gymnostemium or column (Monandrae or

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